Site-directed mutagenesis of evolutionarily conserved serine residues in the N-terminal domain of rice Phi-class glutathione S-transferase F5

Abstract

To identify the critical residue for the catalytic activity of rice glutathione S-transferases (GSTs), the evolutionarily conserved serine residues in the N-terminal domain of rice phi class GST F5 (OsGST F5) were replaced with Ala by site-directed mutagenesis. Replacement of Ser9 with Ala significantly affected the catalytic activity and kinetic parameters of the enzyme, whereas substitutions of Ser34 and Ser65 with Ala showed little effect. The S9A mutant showed a decrease of approximately 90-99% in the specific activities and k(cat) values. From the plot of pH against log(k(cat)/K-m), the pK(a) values for glutathione (GSH) in the wild-type enzyme-GSH complex and the S9A-GSH complex were estimated to be 6.8 and 8.6, respectively. From these results, we suggest that the evolutionarily conserved Ser9 residue in OsGST F5 plays a major role in the catalytic mechanism of rice GST by lowering the enzyme's pKa and enhancing the nucleophilicity of the GSH thiol in the active site. (C) 2014 Elsevier B.V. All rights reserved.